Combination antiretroviral therapy (ART) is effective in inhibiting HIV infection and reducing rates of transmission, while also improving the quality of life and increasing the life expectancy of patients with HIV/AIDS. However, there are significant drawbacks to the antiviral drug therapy, including potential drug resistance, toxic side effects, and cost of daily medication. Moreover, individuals on long-term treatment can have significant cardiac changes, such as, loss of bone density, premature aging as well as other complications; the collective of which are sufficiently significant to consider the development of novel alternative therapies. The proposed study will focus on gene therapy strategies to establish an HIV-resistant immune system. Our strategy involves the expression of multiple anti-HIV genes in hematopoietic stem/progenitor cells (HSPCs) that are the precursors for HIV-susceptible cells. Hence, the overriding goal of this proposal is develop a combinatorial anti-HIV gene therapy that inhibits many aspects of the virus life cycle, from virus entry to viral expression an production, which will improve upon the anti-HIV efficacy, suppress viral mutant escape, and abate cellular toxicity. This goal builds on our prior work, in which we developed and clinically evaluated an anti-HIV lentiviral vector (rHIV7-shI-TAR-CCR5RZ) that expressed three anti-HIV small RNAs, including a short hairpin RNA (shRNA) targeting the tat/rev of HIV-1 mRNA transcripts, a ribozyme targeting CCR5 mRNA, and a nucleolar-localizing TAR decoy, which represented the first successful demonstration of engraftment and long-term maintenance of gene-modified HSPCs in an HIV infected population. In the previous funding period, we extend this approach by developing the MCM7-S1-S2M-S3B- tRNASer-CCR5sh lentiviral vector, which expresses three anti-HIV siRNAs as miRNA mimics from the MCM7 intron and a fourth CCR5 shRNA from a novel tRNASer promoter. In primary cell and humanized mouse studies, we demonstrated that this design is safer and more efficacious that the original combinatorial lentiviral vector, and we are now preparing it for clinical evaluation. In the proposed studies for this project, we will apply this same innovation to develop a new generation of highly potent anti-HIV lentiviral vectors that are designed to be safer, more efficacious, and more resistant to viral escape than any previous generation of anti-HIV vectors and are capable of virus-independent enrichment of vector transduced HSPCs. In Specific Aim 1 we will test combinations of anti-HIV genes for anti HIV activity and resistance to mutational escape in long term T-cell and hematopoietic cell culture. By combining our most potent multi-resistant shRNA, a tRNA-aptamer, and TRIM5? components, we will create novel combinatorial lentiviral vectors, designed to be highly effective, resistant to mutational escape, and nontoxic to cells. In Specific Aim 2, we will optimize vector packaging, transduction, and enrichment of transduced HSPCs. In the pre-clinical development of anti-HIV lentiviral vectors, it is imperative to design vectors that are highly potent against HIV without compromising vector packaging and the feasibility of scale-up. This aim will address these concerns, as well as investigating methods for improving transduction efficiencies, selection of transduced HSPCs. In Specific Aim 3, we will test vector transduced hematopoietic stem cells in a NSG humanized mouse model to evaluate the efficacy, safety, and toxicity of new combinatorial lentiviral vectors in CD34+ HSPCs. Because we are developing combinatorial vectors that incorporate new anti-HIV genes and novel expression strategies, we will utilize the humanize NSG mouse model to address potential concerns regarding the hematopoietic toxicity, interferon activation, antiviral activity, and overal safety. We will also evaluate long-term antiviral protection and resistance to mutational escape for each new vector in mice challenged with R5-tropic HIV-1BaL or R5/X4-tropic HIV-189.6.